OVERALL HYPOTHESIS: Signaling efficiency/specificity for heterotrimeric G-protein systems is determined in part by accessory proteins which regulate the efficiency and/or specificity of signal transfer from G-protein coupled receptors (GPCRs) to G-proteins, segregate a signaling complex to microdomains of the cell, regulate the basal activity of the system and/or provide alternative modes of signal input to G-protein signaling systems that operate independent of a typical GPCR. Our objectives are to define such accessory proteins, their mechanism of regulation, their dysfunction in various diseases and their potential as therapeutic targets. We recently identified a group of proteins (Activators of G-protein signaling (AGS) 1-8 that directly influence the activation state of G-proteins independent of a GPCR. AGS proteins interact with different subunits and/or conformations of heterotrimeric G-proteins and selectively regulate different types of G-proteins. This proposal focuses on AGS1, which is unique among the AGS family members. AGS1 is a Ras related protein that regulates heterotrimeric G- protein signaling providing a surprising mechanism for cross talk between small and large (heterotrimeric) G- protein families. A major goal of this application is to understand how AGS1 integrates into cell and organ function. SPECIFIC AIM #1 Define the subcellular location of AGS 1 and the influence of receptor and signal pathway activation on its subcellular location and nucleotide binding state. SPECIFIC AIM #2 Define the domains of AGS 1 required for interaction with and regulation of heterotrimeric G-proteins. SPECIFIC AIM #3 Define the mechanism by which AGS1 regulates signal processing by GPCRs. SPECIFIC AIM #4 Identify and characterize AGS 1 regulators and additional AGS 1 effectors. AGS1 and related accessory proteins provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers and the cellular functions they regulate. As such, these proteins and the concepts advanced with their discovery provide unexpected avenues for therapeutic development and increased understanding of disease mechanisms.